Purification And Characterization Of The Linoleate Isomerase From Lactobacillus Plantarum ZS2058

Conjugated linoleic acid (CLA) refers to geometric and positional isomers of linoleic acid (LA) with two conjugated double bonds. Originally discovered as a naturally occurring anticarcinogen, CLA has been subsequently show to lower cancer risk, enhance immunity and reduce body fat. Especially the c9, t11-CLA is recognized as the most biologically active isomer. Although methods have been described for the isolation of CLA isomers from chemically synthesized mixtures, the processes are expensive and often do not produce a single isomer at high purity. Some microorganisms such as the Lactobacillus plantarum, are capable of producing CLA from LA. The mechanism is that the existence of linoleate isomerase enzyme convert linoleic acid to CLA. So the study of linoleate isomerase enzyme has been the focus of considerable research efforts in recent years.Lactobacillus plantarum ZS2058, which was screened from the Chinese traditional fermented vegetable, has the capacity to convert the LA into CLA. The location of Lactobacillus plantarum ZS2058 linoleate isomerase and partial characterization of the crude enzyme extract were studied in this paper. The optimum disruption conditions of ultrasonicwave for Lactobacillus plantarum ZS2058 were as follows:power of treatment 400W, time of treatment 7.5min (time for each treatment was 5s, with a 9s interval). After an ultra centrifugation step (150,000×g, 1h, 4℃) to separate membrane from soluble material, 70% of the enzyme activity was detected in the membranous material. And Triton X-100 could increase the enzyme activity in the crude enzyme extract significantly. But NaCl could not increase the crude enzyme activity significantly. These results support the conclusion that Lactobacillus plantarum ZS2058 linoleate isomerase is a membrane-associated protein (integral). The optimum concentration for linoleic acid was determined to be 0.08mg/mL. And 5% glycerol could enhance the stability of the crude enzyme.The optimum saturation of Ammonium sulphate for linoleate isomerase was 30-50%. The anion exchange column was eluted with a NaCl gradient of 0-0.75M, and the linoleate isomerase activity peak was eluted at 0.12M NaCl when the pH value of Tris-HCl buffer was 9.0. The linoleate isomerase was purified by Ammonium sulphate precipitation, anion exchange and size exclusion column chromatography, achieving an overall purification of 8.49-fold and a specific activity of 168.58U/mg. The purified enzyme was a single band of around 66kDa on SDS-PAGE.The optimum temperature for linoleate isomerase activity was about 35℃. And the optimum pH was about 5.5. No external cofactors or energy source were required for catalysis. The effect of the metal-chelators (EDTA and EGTA) and metal ions were not significant. The Km and Vmax for linoleate isomerase were 2.15×10-5mol/L and 3.71×10-9mol/ (min?mg), respectively.